The C-terminal conserved domain of MARCKS is phosphorylated in vivo by proline-directed protein kinase. Application of ion trap mass spectrometry to the determination of protein phosphorylation sites.
MARCKS, the major protein kinase C substrate in various cells and tissues, binds to calmodulin, acidic membrane phospholipids, and actin filaments, and these interactions are regulated by protein phosphorylation. We have previously shown that MARCKS purified from bovine brain is phosphorylated not only by protein kinase C but also by so-called proline-directed protein kinases in the well conserved N-terminal half of the molecule (Taniguchi, H., Manenti, S., Suzuki, M., and Titani, K. (1994) J. Biol. Chem. 269, 18299-18302). Although the presence of other phosphorylation sites in the C-terminal peptide was also noticed, the ambiguity in the C-terminal domain of the bovine protein hampered a more detailed analysis. In the present study, we analyzed MARCKS purified from rat brain by electrospray ionization/ion trap mass spectrometry. The results obtained revealed two additional novel phosphorylation sites in the C-terminal region. Both phosphorylation sites (Ser291 and Ser299) are immediately followed by proline, suggesting that these sites are also phosphorylated by the proline-directed protein kinase(s). Since Ser299 is within the C-terminal domain, which is well conserved among various species, the function of the domain, whatever it is, seems to be controlled by phosphorylation.
Yamauchi
E
E
Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Nagoya City University, Mizuho-ku, Nagoya 467, Japan.
Kiyonami
R
R
Kanai
M
M
Taniguchi
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods; D047908:Intracellular Signaling Peptides and Proteins; D013058:Mass Spectrometry / Q000379:methods; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010766:Phosphorylation; D038461:Proline-Directed Protein Kinases; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D017386:Sequence Homology, Amino Acid
D047908:Intracellular Signaling Peptides and Proteins; C000619712:Marcks protein, rat; D008565:Membrane Proteins; D011506:Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D038461:Proline-Directed Protein Kinases; D017346:Protein Serine-Threonine Kinases